Open AccessResearch article Synthetic xylan-binding modules for mapping of pulp fibres and wood sections Lada Filonova1, Lavinia Cicortas Gunnarsson2,3, Geoffrey Daniel*1 and Address:
Trang 1Open Access
Research article
Synthetic xylan-binding modules for mapping of pulp fibres and
wood sections
Lada Filonova1, Lavinia Cicortas Gunnarsson2,3, Geoffrey Daniel*1 and
Address: 1 WURC, Department of Wood Science, Swedish University of Agricultural Sciences, PO Box 7008, SE-750 07 Uppsala, Sweden,
2 Department of Immunotechnology, Lund University, BMC D13, S-22184 Lund, Sweden and 3 Current address : Affitech AS, Oslo, Norway
Email: Lada Filonova - lada.filonova@trv.slu.se; Lavinia Cicortas Gunnarsson - l.gunnarsson@affitech.com;
Geoffrey Daniel* - geoffrey.daniel@trv.slu.se; Mats Ohlin - mats.ohlin@immun.lth.se
* Corresponding author
Abstract
Background: The complex carbohydrate composition of natural and refined plant material is not
known in detail but a matter that is of both basic and applied importance Qualitative assessment
of complex samples like plant and wood tissues requires the availability of a range of specific probes
Monoclonal antibodies and naturally existing carbohydrate binding modules (CBMs) have been used
in the past to assess the presence of certain carbohydrates in plant tissues However, the number
of natural CBMs is limited and development of carbohydrate-specific antibodies is not always
straightforward We envisage the use of sets of very similar proteins specific for defined targets,
like those developed by molecular evolution of a single CBM scaffold, as a suitable strategy to assess
carbohydrate composition An advantage of using synthetic CBMs lies in the possibility to study fine
details of carbohydrate composition within non-uniform substrates like plant cell walls as made
possible through minor differences in CBM specificity of the variety of binders that can be
developed by genetic engineering
Results: A panel of synthetic xylan-binding CBMs, previously selected from a molecular library
based on the scaffold of CBM4-2 from xylanase Xyn10A of Rhodothermus marinus, was used in this
study The wild type CBM4-2 and evolved modules both showed binding to wood sections
However, differences were observed in the staining patterns suggesting that these modules have
different xylan-binding properties Also the staining stability varied between the CBMs, the most
stable staining being obtained with one (X-2) of the synthetic modules Treatment of wood
materials resulted in altered signal intensities, thereby also demonstrating the potential application
of engineered CBMs as analytical tools for quality assessment of diverse plant material processes
Conclusion: In this study we have demonstrated the usefulness of synthetic xylan-binding modules
as specific probes in analysis of hemicelluloses (xylan) in wood and fibre materials
Background
Wood cell walls represent highly complex biocomposites
that consists of repertoires of polysaccharides and lignins integrated into a three-dimensional conglomerate [1,2]
Published: 12 October 2007
BMC Plant Biology 2007, 7:54 doi:10.1186/1471-2229-7-54
Received: 14 June 2007 Accepted: 12 October 2007
This article is available from: http://www.biomedcentral.com/1471-2229/7/54
© 2007 Filonova et al; licensee BioMed Central Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Trang 2At the microstructural level, wood cell walls are composed
of both primary- and dominating secondary cell wall
lay-ers in which the major (cellulose, lignin and
hemicellu-loses) and minor (pectins, proteins) chemical
components are heterogeneously dispersed and
inte-grated Understanding the cell wall's complexity is of great
interest both for plant biology and for plant processing in
food and pulp industry
One of the key main challenges for an improved
under-standing of wood cell wall structure especially, in relation
to properties, is an ability to specifically localise and
visu-alised the spatial distribution of the major polysaccharide
components (cellulose and hemicelluloses) in vivo In
lig-nified cell walls this is quite challenging since the
hemicel-luloses interface between the cellulose and lignin forming
a heterogeneous polymer complex and it is exacting to
distinguish between the various components in the
differ-ent cell wall layers Xylan is the dominant hemicellulose
found in hardwoods and depending on species, normally
represents 15–30% of the total dry matter content [3]; the
major component being an
O-acetyl-4-O-methylglu-curono-β-D-xylan referred as glucuronoxylan In
soft-woods, xylan normally forms 5–10% of the total dry
matter as arabinoglucuronxylan and consists of a
back-bone of 1,4-linked β-D-xylopyranose units, partially
sub-stituted by 4-O-methyl-α-D-glucuronic acid and
α-L-arabinofuranose units [3] Knowledge on the
microdistri-bution of hemicelluloses (i.e xylans) within wood cell
walls has come primarily from early gross chemical
stud-ies on sections from cambium layers at different stages of
cell wall differentiation [4], by the application of
xyla-nase-gold complexes to plant cell walls and pulp fibres
[5-7] and by microscope observations after selective xylan
removal [8,9] More recently monoclonal antibodies
spe-cific for xylans have been successfully used as probes on
plant fibres (flax) and other non-lignified plant materials
[10], but not wood There has also been great interest to
chemically map and assess the removal and sorption of
xylans onto fibres during kraft pulping processes in order
to develop improved washing and preventive methods
thus improving the technical properties of paper products
[5] However, apart from these examples very little is
known on the microdistribution of xylans across lignified
wood cell walls
A plethora of direct (e.g enzyme-gold) and indirect
meth-ods (e.g antibodies) have been employed to visualise
car-bohydrates in plant materials by both light- and electron
microscopy [11,12] Historically, antibodies have been
the markers of choice and a range of monoclonal probes
have been produced for visualising plant cell wall
carbo-hydrates (e.g xylans, galactans, glucomanans, arabinans
and fucosylated xyloglucan) [10,13,14] More recently,
several non-catalytic carbohydrate binding modules
(CBMs) have also been reported to show great potential as specific and versatile markers of carbohydrates in plant materials [15-19] CBMs are a group of natural binders that most often constitute parts of modular glycoside hydrolysing or modifying enzymes Based on their sequences, CBMs are classified into 49 families http:// http:afmb.cnrs-mrs.fr/CAZY/, however, there are large variations in binding specificities, towards crystalline, amorphous and soluble polysaccharides, both between and within the families [20,21] CBMs represent an attrac-tive alternaattrac-tive to antibody probes not only because of their size (e.g typically < 20 kDa vs ~150 kDa for IgGs) and intrinsic specificity to individual carbohydrates – often different regions of the same substrate may be tar-geted- but also because of their ease of production and ability for modification with peptides and fluorescent (e.g fluorescein isothiocyanate (FITC), tetramethylrhod-amine isothiocyanate (TRITC)) and gold markers for microscopy [15-17,22-24] We have previously developed methods for detection of cellulose in wood cell walls by visualising native cellulose-specific CBMs (i.e CBMPcCel7D, CBM1HjCel7A) bound to their target in both light- and elec-tron microscopy [15-17] Although several natural CBMs are available for these applications, the numbers are rela-tively low It is reasonable to expect that probes showing superior performance in given applications will be availa-ble by having a larger population of molecules to choose from Combinatorial library and selection technologies provide a solution to this need Here we further developed this approach and report on the application of a range of engineered xylan-binding CBMs for targeting xylan in wood (birch and pine) sections and on the surface of birch kraft pulp fibres The advantages of using synthetic CBMs -which is novel for this study- over native CBMs is related to their molecular properties such as stability and binding specificity that can be targeted by engineering The synthetic xylan-binding CBMs utilised in this work for
in-situ visualisation of xylan in fresh wood sections and
whole fibre materials performed well, clearly demonstrat-ing their usefulness in studies on plant materials
Results and discussion
Binding properties of xylan-binding CBMs
The wild type (wt) CBM4-2 from the xylanase Xyn 10A of
Rhodothermis marinus is a type B CBM [20] displaying a
groove-shaped binding site that fits polysaccharide chains well [25] This CBM binds not only to xylan but also other non-crystalline plant carbohydrates [26,27] In a previous study, we had constructed a combinatorial library of CBM4-2 [28] and used the phage-display system to select for CBM-variants specific for different ligands The bind-ing properties of three CBMs, X-2, X-6 and X-13, all selected using the insoluble part of birch wood xylan as a selection target were further investigated in this work with the aim of defining the potential to use such modules in
Trang 3analysis of xylans in plant tissues The sequences of these
proteins and a control module unable to bind plant
car-bohydrates are all very similar and they differ from the wt
protein (of 167 residues) by only 4–9 amino acid
substi-tutions (Figure 1) Despite the fact that they originate
from the same selection, these CBMs have different
affin-ities and specificaffin-ities for a number of tested
carbohy-drates Affinity electrophoresis demonstrated that the X-6
variant, like wt CBM4-2, binds to a range of carbohydrates
(Figure 2) In contrast, both X-2 and X-13 are substantially
more specific for xylans and do not recognise xyloglucan
or glucan-containing carbohydrates (Figure 2) In
addi-tion, isothermal titration calorimetry studies further
con-firmed that X-2 was much more prone to bind xylans than
any other carbohydrate targets investigated further
dem-onstrating its modified specificity [27] Relative binding
affinities, determined by affinity electrophoresis, revealed
a decrease in affinity for xylan of X-2 and X-13 and a
somewhat higher affinity for xylan of X-6 compared to the
wt CBM4-2 (Table 1) Altogether we have demonstrated
our access to a range of very sequence-similar CBMs with
diverse binding properties that can be used as a utility in
analysis of the carbohydrate composition in
lignocellu-lose materials
Binding of the CBMs to untreated wood sections
In order to assess the ability of the xylan-binding modules
to function as analytical probes, we used these proteins for
staining tissue samples derived from three wood species
known to differ in their content of xylan A very high
con-tent of xylan-based carbohydrates is characteristic for
Bet-ula verrucosa (birch) wood [29,30] Pinus sylvestris (pine)
wood contains approximately 20% of the xylan found in
birch wood [31,32], while wood derived from Populus
tremuloides (poplar) is intermediate [33-35] Moreover
birch and poplar tension wood, but not pine wood,
con-tains fibres with a special morphology and chemical
com-position due to development of a gelatinous layer
(G-layer) [36,37] The G-layer is known to have high cellulose
content with a high degree of crystallinity [34,38,39] We
assume that the combination of almost pure cellulose in
the G-layer and hemicelluloses in S1, S2 and S3 layers
make tension wood an interesting model for assessment
of carbohydrate specific probes We therefore assessed the xylan-binding CBMs that displayed different xylan-bind-ing properties, as outlined above in these models Fluorescent microscopy demonstrated substantial differ-ences in staining patterns of birch, poplar and pine wood sections with wt CBM4-2, X-2, X-6 and X-13 (Figure 3) All CBMs showed binding in comparison to the control sam-ples (Figure 3A – C), wt CBM4-2 and X-13 displaying the most efficient wood labelling (Figure 3D – F; M – O) Although less intense, more stable staining was character-istic for X-2 staining experiments (Figure 3G – I), where the same intensity of fluorescent signal was retained for at least 72 hours giving this module an important advantage
as a specific binding probe in assay setups that demand higher labelling stability The observed (Figure 2, Table 1) reduced affinity for xylan of some of the engineered mod-ules, in particular X-13, obviously did not prevent their use in these applications The binding of lower affinity CBMs is likely to be facilitated by high local concentration
of repetitive structures recognised by them in plant tis-sues Alternatively, the affinity of e.g X-13 for some spe-cific structures in xylan may be higher than that observed when assessing the affinity for solubilized carbohydrates allowing for its use in staining of plant materials
In birch and poplar samples, wt CBM4-2 and X-6 bound
to the cell wall but predominantly to G-layers reported to consist of almost pure cellulose (Figure 3D, F, J, L) whereas X-2 and X-13 signals were detected within S1, S2 layers and middle lamellar regions (Figure 3G, I, M, O) These differential staining patterns are best explained by different affinity and probably more importantly specifi-city profiles of the CBMs, as outlined above Given the inability of these modules to bind crystalline cellulose, it
is possible that wt CBM4-2 and X-6 CBMs labelled xyloglucans within the G-layer, possibly in areas with residual lignin [40] To define in more detail the presence
of binding sites recognised by these xylan-binding CBMs,
it was observed that all CBMs, i.e both those that are (wt
CBM4-2 and X-6) and those that are not (X-2 and X-13) cross-reactive to other types of hemicellulose carbohy-drates like xyloglucans, stained pine samples in areas of
Sequence differences between the five different modules
Figure 1
Sequence differences between the five different modules Each protein is 165 amino acid residues long not counting
the hexa-histidine tag, used in this investigation Amino acids are represented by the standard one-letter code Identities with the wt CBM4-2 sequence are shown by dots Residue numbering is in accordance with Simpson et al [25]
Trang 4bordered pit chambers (including torus) in earlywood
and on the inner surface of cells in latewood (Figure 3E,
H, K and 3N) There is however no previous evidence for
the existence of xyloglucans in bordered pit chambers It
has been shown previously using selective enzyme
removal and enzyme-antibody labelling that bordered pit
membranes in pine sapwood tracheids are composed of
non-lignified pectin and a cellulose-rich region in the
cen-tre of the pit called a torus, which is supported by a
sur-rounding margo that contains microfibrils of cellulose
[41,42] The fact that modules that are quite specific for
xylose oligomers bind to these structures, suggests
pres-ence of at least rudimentary xylan in pine bordered pit
chambers
Variation in binding patterns of the different
CBM-vari-ants to xylan embedded in the wood cell walls was also
observed in the study of McCartney et al [18] where some
natural xylan-binding CBMs stained both primary and
secondary cell walls while some (including CBM4-2) only
stained secondary walls Apart from the CBM's specificity
and the actual presence of particular xylan structures,
lig-and accessibility is also a determinant factor for specific
staining of plant materials using this type of modules The
binding mechanism of many xylan-binding CBMs involves sandwiching of the ligand, which then requires access to both faces of the carbohydrate chain that may be
a limitation in lignocellulose materials It is believed that the X-2 module has evolved to use only one aromatic res-idue for interaction with only one face of the xylan chain [27] These binding features of X-2 could facilitate its binding to the embedded xylan chains and may explain the very stable X-2 staining in the present study Alto-gether, the availability of following a molecular evolution procedure of sets of CBMs with defined binding proper-ties, some members of which are less cross-reactive in comparison to naturally occurring CBMs [27], allow assessment and profiling of carbohydrate composition of plant cell walls and define differences observable between various samples
Binding of the CBMs to treated wood sections and pulp fibres
As outlined above, CBMs derived from the library built on the CBM4-2 scaffold [28] can be used to investigate native and normal wood tissues Modification of such material
by different means may add additional information con-cerning their construction To exploit these possibilities,
we investigated the ability of X-2 to recognise xylan mole-cules in birch and pine wood samples modified through biotechnological processing
Staining of delignified tissue samples (Figure 4I–L) with FITC-conjugated X-2 resulted in much higher signals in comparison to staining of mature (normal) birch and pine tissues (Figure 4E – H) In normal pine sections, X-2 binding was mainly detected in border pit regions (early-wood) and inner surface of cells (late(early-wood) (Figure 4E, F)
Affinity electrophoresis of soluble CBM-variants
Figure 2
Affinity electrophoresis of soluble CBM-variants Affinity electrophoresis performed in the absence of a ligand (A) or in
the presence of 0.2% (w/v) oat spelt xylan (B), 0.1% (w/v) arabinoxylan (C), 0.05% (w/v) barley β-glucan (D), 0.1% (w/v) lichenan (E) or 0.05% (w/v) non-fucosylated xyloglucan (F) Samples included: lane 1, Kaleidoscope prestained standard; lane 2,
wt CBM4-2; lane 3, X-2; lane 4, X-6; lane 5, X-13
Table 1: Affinities of CBM-variants for birch wood xylanas
determined by affinity electrophoresis
Trang 5whereas in birch section staining was observed mostly in
middle lamellae regions (Figure 4G, H) In contrast,
sig-nals from X-2 binding were evenly distributed over the
surfaces of delignified sections in both wood types (Figure
4I – L) This finding suggests, as expected, that removal of
lignin increases the access to the xylan binding sites
Stain-ing of delignified samples by various CBMs may be
bene-ficially preformed to ensure the efficiency of the
delignification procedure and to exploit the access of
spe-cific carbohydrate epitopes prior to enzyme treatment
(high cost procedures) of wood materials Similarly,
hemicellulose removal following a bleaching procedure
could be efficiently assessed in pulp fibres (Figure 5)
fur-ther expanding the area of useful applications of these molecularly engineered modules
Conclusion
In this study, we have demonstrated that molecularly engineered CBMs based on the CBM4-2 scaffold can have
a wide range of applications in basic wood research We envisage that such engineered modules will provide the research community with powerful new tools to comple-ment existing detection reagents such as monoclonal anti-bodies or natural CBMs In addition to the xylan-specific CBMs used in this work, we have also reported the suc-cessful selection of CBM-variants from the same molecu-lar library for their specificities towards other plant cell
Binding of CBMs to different wood sections
Figure 3
Binding of CBMs to different wood sections Binding of wt CBM4-2 (D-F), X-2 (G-I), X-6 (J-L) and X-13 (M-O) to birch,
pine and poplar wood sections Negative control staining of wood samples with mouse anti-His antibody and FITC-conjugated anti-mouse IgG is shown in (A-C) All modules bind to border pit chambers in earlywood and to the latewood lumen wall in pine (blue arrows) Pink arrows show staining of G-layers with wt CBM4-2 and X-6 in birch and poplar cell walls (D, F, J, L), a staining that was not observed with X-2 and X-13 (G, I, M, O; white arrowheads) Note all images are from transverse sections and images of pine sections include examples of both early- (above) and latewood (below) Scale bar = 50 μm
Trang 6wall carbohydrates, such as mannan and xyloglucan
[28,43] The use of engineered CBMs in future studies will
further expand the potential of these types of synthetic
probes for mapping of pulp fibre and wood sections The
binding properties of these modules would also make
them useful in the analysis of technological processes
including those relevant for quality assessment of
indus-trial processes
Methods
Production and purification of CBMs
Protein variants evolved from the scaffold of CBM4-2,
from the xylanase 10A of Rhodothermus marinus, were
selected in a previous study [28] for binding to birch
wood xylan or binding to a human IgG4 molecule Three
CBM-variants from the selection towards xylan (X-2, X-6
and X-13), the wt CBM4-2 and a human IgG4-specific
module (G-4) that served as a non-carbohydrate binding control for the scaffold, were used in this work Genes coding for the proteins with a C-terminal His6-tag had previously been cloned in the pET-22b(+) vector (Nova-gen, Madison, WI, USA) and then transformed in the
Escherichia coli strain BL21(DE3) (Novagen) These genes
were now expressed and the soluble CBMs were purified using metal-ion-affinity chromatography as described ear-lier [26,28]
FITC-labelling of CBMs
One of the xylan-specific CBMs, X-2, and the control module G-4 were labelled with fluorochrome This label-ling is facilitated by the fact that these, as well as most other variants selected from this combinatorial library, carries only two primary amino groups, both of which are located well away from the carbohydrate binding surface
Binding of FITC-conjugated X-2 to wood samples
Figure 4
Binding of FITC-conjugated X-2 to wood samples Pine and birch sections stained with either FITC-conjugated G-4
(A-D) (negative control) or with FITC-conjugated X-2 (E-L) Sections were either untreated (A-H) or delignified (I-L) prior to staining Pink arrows show signals in untreated samples Note that A, C, E, G, I and K are from transverse sections and B, D, F,
H, J and L are from longitudinal sections Pine sections in A and E show both early- (to the left) and latewood (to the right) Scale bar = 50 μm
Trang 7FITC was dissolved in dried N, N-dimethyl formamide
(DMF) to a concentration of 1 mg/ml The purified CBMs,
stored in 20 mM NaH2PO4 (pH 7.5), were first diluted 1:1
in 0.2 M Na2CO3 (pH 9) and FITC was slowly added to
reach 10-fold molar excess relative to CBM The reaction
mixture was gently stirred in a glass vial for 1 hour at room
temperature and then at 4°C overnight NH4Cl was added
to a final concentration of 50 mM in order to inactivate
the un-reacted FITC molecules and stirring was continued for 3 hours CBM-FITC conjugates were separated from FITC by using PD-10 desalting columns (Amersham Bio-science, Uppsala, Sweden) and eluting with 20 mM NaH2PO4 (pH 7.5) Several fractions were collected dur-ing elution and those with the highest absorbance at 280
nm were pooled together The actual labelling ratios 1.6 (X-2) and 0.6 (G-4) mol fluorochrome/mol protein were
Binding of FITC-conjugated X-2 to birch pulp
Figure 5
Binding of FITC-conjugated X-2 to birch pulp Control staining of fibres with FITC-conjugated G-4 is shown in A
(unbleached birch pulp) and B (unbleached birch pulp, after hemicelluloses removal) Binding of X-2 to unbleached pulp fibres was mostly observed at the sites of fibre damage (C), whereas bleached birch pulp were more uniformly stained (E) like the wood sections in Figure 4 Removal of hemicelluloses from both pulp types reduced the fluorescent signal (D, F) proving the specificity of X-2 for xylan It was possible to treat pulps until complete removal of xylan recognised by X-2 was obtained Scale bars = 100 mm for A – D, 200 mm for E, F
Trang 8determined spectrophotometrically by measuring the
absorbance at 280 nm and 495 nm and using the
extinc-tion coefficient 195 for FITC at 495 nm
Affinity electrophoresis
Affinity electrophoresis was performed using the Bio-Rad
(Hercules, CA, USA) mini-gel system as previously
described [26] Purified CBMs (3 μg per gel) were
sepa-rated at room temperature at 90 V on native gels,
contain-ing none or different concentrations of oat spelt xylan
(Sigma-Aldrich, St Louis, MO, USA), birch wood xylan
(Roth, Karlsruhe, Germany), arabinoxylan (Megazyme,
Bray, Ireland), barley β-glucan (Megazyme), lichenan
(Sigma-Aldrich) and tamarind seed xyloglucan
(Meg-azyme) A kaleidoscope pre-stained standard (Bio-Rad)
was included in each gel and proteins were detected by
staining with Coomassie Brilliant blue or Simply Blue
Safe stain (Invitrogen, Paisley, UK) Affinity constants for
birch wood xylan were calculated according to the theory
of affinity electrophoresis as described by Takeo [44] after
performing a series of runs on gels with a ligand
concen-tration ranging from 0.2 to 2 g/l The relative mobility of
a CBM-variant, r (in presence of ligand) and R (in absence
of ligand), versus BSA used as a negative, non-interacting
control were calculated from the migration distances, the
distances between the major protein band and the
migra-tion front of the gel The -1/KD value was obtained as the
x-intercept of a straight line in a plot of 1/(R-r) versus 1/c
according to the relationship:
1/(R-r) = (1 + KD/c)/R where c is the ligand concentration in the gel
Preparation of plant materials
Serial wood sections (15 μm) from poplar (Populus
tremu-loides), birch (Betula verrucosa Ehrh.) and pine (Pinus
sylves-tris L.) were prepared using a Leitz microtome Mild
delignification was in some cases performed on semi-thin
sections of pine and birch using a 1:1 (v/v) mixture of
H2O2 and concentrated acetic acid prior to labelling
Alter-nate sections from the same wood specimens were used
for each labelling experiment
Oxygen bleached and unbleached birch pulp fibres (kindly provided by Eka Chemicals) both with and with-out hemicellulose removal (Table 2) were used for bind-ing experiments with CBM-FITC Hemicellulose removal was performed by treatment of pulp with 24% (w/v) potassium hydrochloride at room temperature for 6 h (shaking) followed by triple washing (20 min each, shak-ing) Thereafter pulps were treated with 17.5% sodium hydrochloride (w/v) containing 4% (w/v) boric acid at room temperature for overnight
Binding of xylan-binding CBMs to wood sections and pulp fibres
Binding experiments on wood sections using all four xylan-binding CBMs (wt CBM4-2, X-2, X-6 and X-13) were performed as follows An initial blocking step was carried out, for preventing any non-specific binding of CBMs, by incubating all samples in phosphate-buffered saline (PBS) (pH 7.4) containing 5% (w/v) ovalbumin Wood samples (2 alternate sections per CBM-variant) were then incubated in 500 μl of PBS containing 10 μM CBM at room temperature for 6 h Thereafter the samples were washed twice with PBS (5 minutes each step) and incubated with mouse Anti-His antibody (Amersham) at 4°C overnight After washing three times with PBS (10 minutes each step), samples were treated with FITC-con-jugated anti-mouse IgG (Sigma) diluted 1:500 in PBS for
1 h in room temperature As negative control, wood sec-tions were incubated with both antibodies
In addition, wood sections, untreated or delignified, as well as bleached or unbleached birch pulp fibres (a con-stant dry weight of 2 mg) were incubated in 500 μl of PBS containing 2 μM FITC-conjugated X-2 and G-4 for 4 h at room temperature followed by washing in PBS Since G-4,
a module selected from the same library as the xylan-binding CBMs, had been shown unable to bind xylan and other hemicelluloses [28], its FITC-labelled derivative served as a negative control in these experiments
In order to detect the bound CBM molecules (FITC-conju-gated or by using labelled antibodies), the samples were placed on object glasses mounted in Fluorsave (Calbio-chem, CA, USA), covered by cover slips and examined by fluorescent microscopy (Leica, Germany) using a standard
Table 2: Characteristics of the birch pulps used for the labelling experiments
Trang 9set of filters for FITC Images were recorded via a CCD
camera (Leica DC 300F, Microsystem) using a digital
imaging system for professional microscopy (Leica
Microsystem Ltd, 2001) with one exposure time (2 s) for
all samples in order to detect differences in signal
inten-sity
Gene sequences
Sequences of protein modules used in this work are
avail-able from public databases as follows: CBM4-2:
[Gen-Bank:AAT06926], X-2: [GenBank:AAT06999], X-6:
[GenBank:AAT07002], X-13: [GenBank:AAT07003] and
G-4: [GenBank:AAT07011]
Abbreviations
CBM, carbohydrate binding module; FITC, fluorescein
isothiocyanate; PBS, phosphate-buffered saline; wt, wild
type
Competing interests
The author(s) declares that there are no competing
inter-ests
Authors' contributions
LF carried out the bio-analytical study involving the
fluo-rescence microscopy and helped to draft the manuscript
LCG carried out the characterisation of the previously
engineered proteins as well as FITC-labelling of the
pro-teins and helped to draft the manuscript GD participated
in the coordination and design of the bio-analytical study
and helped to draft the manuscript MO participated in
the coordination and design of the molecular engineering
and protein characterisation studies and helped to draft
the manuscript All authors read and approved the final
manuscript
Acknowledgements
We gratefully acknowledge the financial support from the Swedish
Research Council, the Wallenberg Foundation (GD) and the Formas
Func-fiber Centre (LF, GD).
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